DESCRIPTION: Ion channel regulation within neuronal and muscle membranes is an important determinant of electrical excitability in the nervous and cardiovascular systems, skeletal muscle, GI tract, and uterus. Voltage-gated K+ channels (Kv channels) play an important role in setting the resting potential and determining repolarization in these functionally diverse systems. Recent evidence suggests that specialized microdomains commonly referred to as lipid rafts exist within the plane of most plasma membranes. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction molecules. In the Preliminary Data section, we demonstrate that the voltage-gated K+ channels, Kv2.1, Kv1.1, Kv1.5, and Kv1.4, but not Kv4.2, target to lipid rafts in both heterologous expression systems and rat brain. In addition, Kv2.1 and Kv1.5 probably reside in different raft compartments. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1-associated rafts and shifts the midpoint of Kv2.1 inactivation by 30-40 mV without affecting peak current density or channel activation. Incubation of Kv2.1 expressing cells with fumonisin B, an inhibitor of ceramide synthase, causes a similar shift in the inactivation curve. Ceramide is both a raft component and an intracellular signaling molecule. Thus, raft association is functionally significant, for such a shift in the inactivation will result in a large percentage of the Kv2.1 channels being functionally silenced in the range of physiological membrane potentials. In addition, the preliminary data suggest fumonisin B induces mistargeting of Kv2.1 from the cell body to the distal dendrites in cultured neurons, suggesting raft-related signaling mechanisms are involved in Kv2.1 targeting. The Specific Aims will 1) address the mechanisms involved in the targeting of Kv2.1 to lipid rafts, with emphasis placed on subunit composition and channel transmembrane domains; 2) examine the functional significance of Kv2.1 association with lipid raft domains with emphasis on ceramide signaling pathways, 3) examine the relationship between raft association, ceramide signaling, and cell surface localization in neurons; and 4) begin initial work aimed at purification of Kv 2.1-containing lipid rafts from brain. This proposed research examines a new area in Kv channel research that will have important implications in multiple tissue systems.